This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ribosome is the universal site of protein synthesis, containing some of the most highly conserved of all biological sequences. Nevertheless, the ribosome is robust to mutation, capable of functioning when chal-lenged with base or amino acid substitutions in its highly conserved functional centers. As major targets of antibiotics, these functional centers are the sites of numerous antibiotic-resistance mutations. While it has been well established that antibiotic-resistance mutations carry a substantial fitness cost, the structural basis for this burden is only now within the scope of our technical ability to investigate. Here, we will use X-ray crystallography of ribosomes from the thermophilic bacterium Thermus thermophilus to address the structural robustness of ribosome active sites and its relationship to biological fitness. In collaboration with Albert Dahl-berg and Steven Gregory at Brown University, we will investigate streptomycin-resistance mutations to ex-amine the mutational robustness of a conserved ribosome functional center that participates in global con-formational changes of the 30S subunit. In collaboration with Frank Murphy, we have already collected dif-fraction data sets for 30S subunit crystals carrying streptomycin-resistance mutations and the streptomycin-dependence mutation G524U. Overall, we have crystals available for 30S subunits carrying several antibi-otic-resistance mutations and we are in the process of preparing a large number of additional ribosome sam-ples. In addition to providing a more complete mechanistic understanding of antibiotic resistance at an un-precedented level of resolution, these efforts are directed towards establishing fundamental principles of ri-bosome structural organization and evolution.